The objective of these studies is to analyze the ontogenetic development and subsequent regulation of B cell diversity. This objective will be sought by determination of early V region gene expression, idiotype anti-idiotype interactions and subsequent effects of environmental antigens on B cells. Cell hybridization will be used to isolate cloned lines representative of fusion products of pre-B cells. These cell lines will be used to analyze cellular mechanisms involved in the expression of V genes and immunoglobulin synthesis by direct methodology at a stage before extrinsic antigen can select antibody repertoires. Later stages of B cell development will be analyzed by clonal assays, immunofluorescence and the use of anti-idiotype antibodies as serological probes. The effects of idiotype and anti-idiotype interactions on establishment of the antibody repertoire will be analyzed by direct examination of the interacting cells involved using appropriate markers to each cellular element, combined with the immortalization of idiotypic and anti-idiotypic cells using B cell hybridization. Generation of V region diversity within clones of antigen stimulated B cells will be analyzed using panels of monoclonal anti-idiotype antibodies. The information learned by detailed analyses of idiotypy in the response to Alpha l-3 dextran will be applied to in vivo studies on the role of these antibodies in protection against enteric microorganisms. By using a combination of (i) cell hybridization to isolate B cell clones expressing idiotype and anti-idiotype activity, (ii) clonal assays to investigate the immunoglobulin synthesizing potential of clonal precursors, and (iii) the analysis of responses to bacterial antigens it is planned to integrate basic studies on B cell differentiation with a detailed analysis of the immune response to naturally occurring enteric organisms.
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